猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效
Summary
我们描述了使用猪角膜来测试实验药物的抗病毒功效。
Abstract
病毒和细菌可通过角膜感染导致各种眼部表面缺陷和退化，如伤口和溃疡。由于血清超常范围为全球 60-90%，单纯疱疹病毒类型-1 （HSV-1） 通常会导致或非酸性区域的粘皮病变，这也表现为病变和感染相关失明。虽然目前的抗病毒药物是有效的，但耐药性和持久性毒副作用的出现需要开发针对这种无处不在的病原体的新型抗病毒药物。虽然体外评估提供了一些有关新兴抗病毒药物的功能数据，但它们并不显示体内眼组织具有复杂性。然而，在体内研究是昂贵的，需要训练有素的人员，尤其是当与病毒剂工作。因此，前体内模型是高效但廉价的抗病毒测试步骤。在这里，我们讨论一个协议，研究感染HSV-1使用猪角膜前体内和方法，以治疗他们使用现有的和新的抗病毒药物。我们还演示了使用 HSV-1 进行斑块检测的方法。详细的方法可用于进行类似的实验，以研究类似于HSV-1病原体的感染。
Introduction
患有眼部感染的人经常会出现视力丧失1。由于全球血清高超常，HSV感染者患有复发性眼部感染，导致角膜疤痕、频闪角膜炎和新血管化2、3、4、5。HSV感染也显示导致较少的频率，免疫功能低下，未经治疗的患者，如脑炎和系统性发病率6，7，8的一系列严重情况。像Acyclovir（ACV）及其核苷类比这样的药物在抑制HSV-1感染甚至控制重新激活方面已经取得了一致的成功，然而这些药物的长期使用与肾衰竭、胎儿畸形和未能限制抗药性对进化的病毒株9、10、11、12、13的产生有关。与HSV-1眼部感染相关的复杂性，以前曾使用人类角膜细胞的单层和3D培养物进行体外研究，并使用穆林或兔眼感染进行体内研究。虽然这些体外模型提供了关于HSV-1感染的细胞生物成分的重要数据，但是，它们不能模仿角膜组织的复杂复杂性，也无助于照亮病毒的树突传播。相比之下，虽然体内系统在显示 HSV-1 感染期间角膜感染传播和免疫激活反应方面更有见地，但它们确实附带了警告，即它们需要训练有素的研究人员和大型动物护理设施来忽略实验。
在这里，我们使用猪角膜作为前体内模型来检查HSV-1感染诱发的伤口系统。通过组织外植培养，可以研究某些药物的潜在药理学以及感染引起的伤口系统的细胞和分子生物学。该模型也可以修改为用于其他病毒和细菌感染。在这项研究中，猪角膜被用来测试临床前小分子BX795的抗病毒功效。由于容易获得和成本效益，更倾向于使用猪角膜。此外，猪角膜模型是人眼的良好模型，角膜易于分离，适合感染和可视化，坚固耐用，可处理15。猪角膜也可与人类角膜模型在跨角膜渗透性和全身吸收15的复杂性相媲美。通过使用这个模型进行研究，我们能够阐明BX795作为HSV-1病毒感染的称职抑制剂如何值得进一步研究，并增加了将其分类为潜在的小分子抗病毒化合物16的文献。
Protocol
Representative Results
Discussion
先前的研究表明，BX795作为抗HSV-1感染的抗病毒药物具有很有前途的作用：通过抑制坦克绑定激酶1（TBK1）16。TBK1和自噬在帮助抑制HSV-1感染方面都起到了一定的作用，这表现在人类角膜上皮细胞上。BX795在浓度为10μM的抗病毒活性方面被证明是最大的有效，并且使用西方斑点分析和关键病毒蛋白的病毒斑块分析，BX795被证明可以抑制HSV-1感染，与TFT16的活动相当。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了国家卫生研究院向D.S.A.提供的F30EY024710、RO1 AI139768和RO1 EY029426的资助，并得到了国家眼科研究所F30EY025981赠款的支持。研究使用从公园包装公司获得的猪角膜进行，4107阿什兰大道，新城，芝加哥，IL-60609
Materials
| 30 G hypodermic needles. | BD | 305128 | |
| 500 mL glass bottle. | Thomas Scientific | 844027 | |
| Antimycotic and Antibiotic (AA) | GIBCO | 15240096 | Aliquot into 5 mL tubes and keep frozen until use |
| Benchtop vortexer. | BioDot | BDVM-3200 | |
| Biosafety cabinet with a Bio-Safety Level-2 (BSL-2) certification. | Thermofisher Scientific | Herasafe 2030i | |
| Calgiswab 6" Sterile Calcium Alginate Standard Swabs. | Puritan | 22029501 | |
| Cell scraper – 25 cm | Biologix BE | 70-1180 70-1250 | |
| Crystal violet | Sigma Aldrich | C6158 | Store the powder in a dark place |
| Dulbecco’s modified Eagle’s medium – DMEM | GIBCO | 41966029 | Store at 4 °C until use |
| Ethanol | Sigma Aldrich | E7023 | |
| Fetal bovine serum -FBS | Sigma Aldrich | F2442 | Aliquot into 50 mL tubes and keep frozen until use |
| Flat edged tweezers – 2. | Harward Instruments | 72-8595 | |
| Freezers –80 °C. – | Thermofisher Scientific | 13 100 790 | |
| Fresh box of blades. | Thomas Scientific | TE05091 | |
| Guaze | Johnson & Johnson | 108 square inch folder 12 ply | |
| HSV-1 17GFP | grown in house | – | Original strain from Dr. Patricia Spears, Northwestern University. GFP expressing HSV-1 strain 17 |
| Insulin, Transferrin, Selenium – ITS | GIBCO | 41400045 | Aliquot into 5 mL tubes and keep frozen until use |
| Magnetic stirrer. | Thomas Scientific | H3710-HS | |
| Metallic Scissors. | Harward Instruments | 72-8400 | |
| Micropipettes 1 to 1000 µL. | Thomas Scientific | 1159M37 | |
| Minimum Essential Medium – MEM | GIBCO | 11095080 | Store at 4 °C until use |
| OptiMEM | GIBCO | 31985047 | Store at 4 °C until use |
| Penicillin/streptomycin. | GIBCO | 15140148 | Aliquot into 5 mL tubes and keep frozen until use |
| Phosphate Buffer Saline -PBS | GIBCO | 10010072 | Store at room temperature |
| Porcine Corneas | Park Packaging Co., Chicago, IL | 0 | Special order by request |
| Procedure bench covers – as needed. | Thermofisher Scientific | S42400 | |
| Serological Pipettes | Thomas Scientific | P7132, P7127, P7128, P7129, P7137 | |
| Serological Pipetting equipment. | Thomas Scientific | Ezpette Pro | |
| Stereoscope | Carl Zeiss | SteREO Discovery V20 | |
| Stirring magnet. | Thomas Scientific | F37120 | |
| Tissue culture flasks, T175 cm2. | Thomas Scientific | T1275 | |
| Tissue culture incubators which can maintain 5% CO2 and 37 °C temperature. | Thermofisher Scientific | Forma 50145523 | |
| Tissue culture treated plates (6-well). | Thomas Scientific | T1006 | |
| Trypsin-EDTA (0.05%), phenol red | GIBCO | 25-300-062 | Aliquot into 10 mL tubes and keep frozen until use |
| Vero cells | American Type Culture Collection ATCC | CRL-1586 |
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